Careful structural analysis of the DNA content of several early passage and laboratory adapted strains of Borrelia burgdorferi has revealed, in addition to an approximately 1000 kilobase (kb) genome, a unique collection of terminally crosslinked, linear and covalently closed, circular DNA molecules ranging in size from 1.5 to 50 kb in length. Often DNA contour length profiles appear to differ among isolates and to vary during laboratory passage. Since accurate structural characterization by agarose gel electrophoresis alone is difficult, nucleic acid electron microscopy techniques were used extensively in these studies to assess molecular form, sequence arrangement and genetic relatedness. Each of the linear molecules was shown to rapidly reanneal to linear duplexes after alkaline denaturation, and to form single-stranded, circular molecules measuring twice the length of the linears upon treatment with a combination of methyl mercury, glyoxal, urea and heat prior to mounting for electron microscopy. Both circle formation and rapid reannealing could be prevented by brief pretreatment with single-strand specific nucleases. Furthermore, after enzymatic removal of the terminal crosslink, separable single-stranded molecules could then be compared directly by conventional heteroduplex analysis techniques. Partial denaturation of either intact or single-strand specific nuclease treated molecules showed profiles suggesting that the highest G+C content in each of the linear molecules examined was located near the molecular termini. However, heteroduplex analysis of enzymatically nicked linears revealed little or no inter-strand interaction. A technique was developed for the separation of terminal restriction fragments away from internal fragments using alkali denaturation, followed by a very brief incubation period to allow for concentration independent reannealing and final purification of snap-back, terminal fragments by ion exchange column chromatography. The purified terminal fragments were futher characterized and found to resemble human telomeres in molecular structure.